Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration-and UV-dependent photoincorporation of [ 3 H]atRA into PKC␣, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKC␣, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca 2؉ . At pharmacological concentrations (10 M), atRA decreased PKC␣ activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca 2؉ . These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKC␣ and prevent PKC␣ activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.
It is suggested that formation of more polar metabolites of all-trans-retinoic acid (atRA) via oxidative pathways limits its biological activity. In this report, we investigated the biotransformation of oxidized products of atRA via glucuronidation. For this purpose, we synthesized 4-hydroxy-RA (4-OH-RA) in radioactive and nonradioactive form, 4-hydroxy-retinyl acetate (4-OHRAc), and 5,6-epoxy-RA, all of which are major products of atRA oxidation. Glucuronidation of these retinoids by human liver microsomes and human recombinant UDPglucuronosyltransferases (UGTs) was characterized and compared with the glucuronidation of atRA. The human liver microsomes glucuronidated 4-OH-RA and 4-OHRAc with 6-and 3-fold higher activity than atRA, respectively. Analysis of the glucuronidation products showed that the hydroxyl-linked glucuronides of 4-OH-RA and 4-OH-RAc were the major products, as opposed to the formation of the carboxyl-linked glucuronide with atRA, 4-oxo-RA, and 5,6-epoxy-RA. We have also determined that human recombinant UGT2B7 can glucuronidate atRA, 4-OH-RA, and 4-OH-RAc with activities similar to those found in human liver microsomes. We therefore postulate that this human isoenzyme, which is expressed in human liver, kidney, and intestine, plays a key role in the biological fate of atRA. We also propose that atRA induces its own oxidative metabolism via a cytochrome P450 (CYP26) and is further biotransformed into glucuronides via UGT-mediated pathways. atRA 1 is a major metabolite of vitamin A (all-trans-retinol) that undergoes isomerization and metabolism in vivo, yielding 13-cis-retinoic acid (13-cis-RA), 9-cis-retinoic acid (9-cis-RA)
4-Hydroxy-2-nonenal (HNE), a major electrophilic byproduct of lipid peroxidation, is mutagenic and cytotoxic. The two pairs of HNE-derived diastereomeric 1,N2-propanodeoxyguanosine 3'-monophosphate adducts were synthesized from reaction of HNE with 2'-deoxyguanosine 3'-monophosphate. After HPLC separation, these adducts were characterized by UV-visible absorption and negative ion electrospray ionization MS/MS analysis. To further characterize the structures, these adducts were dephosphorylated to the corresponding HNE-modified deoxyguanosine adducts and their HPLC retention times and UV spectra were compared with those of the synthetic standards prepared from reaction of HNE with 2'-deoxyguanosine. Separation of these adducts by 32P-postlabeling/HPLC was developed. Reaction of HNE with calf thymus DNA resulted in only one pair of diastereomeric adducts, with one adduct predominantly formed with a modification level of 1.2 +/- 0.5 adducts/10(7) nucleotides.
The hepatotoxicity of acetaminophen is believed to be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine; however, the mechanism by which this metabolite produces the toxicity is unknown. The metabolite, which is both an electrophile and an oxidizing agent, may covalently bind to critical proteins, or it may initiate oxidative damage. We have previously developed a Western blot assay for detection of acetaminophen covalently bound to protein and have reported the relationship between covalent binding and the development of hepatotoxicity. Recently, we developed a Western blot assay for protein aldehyde formation, which may occur via the reactive oxygen species, the hydroxyl radical. In this paper, we have compared covalent binding to protein aldehyde formation. Toxic doses of acetaminophen (400 mg/kg) were administered to mice, and the mice were subsequently killed at 0, 1, 2, 4, and 6 h. Since the oxidizing agent FeSO4 has been reported to potentiate lipid peroxidation when administered with acetaminophen, other mice received FeSO4 (100 mg/kg) plus acetaminophen. Compared to saline-treated control mice, acetaminophen treatment significantly increased serum alanine aminotransferase levels, an index of hepatotoxicity, at 4 and 6 h, but not at 1 or 2 h. Acetaminophen plus FeSO4 treatment of mice significantly increased serum alanine aminotransferase levels at 2, 4, and 6 h compared to controls. Levels of alanine aminotransferase in serum of acetaminophen plus ferrous sulfate-treated mice were higher at 4 and 6 h than those of acetaminophen-treated mice, but not significantly different. FeSO4 alone did not increase alanine aminotransferase levels. Western blot assays revealed that acetaminophen did not cause an increase in protein aldehydes over control at any time, nor did acetaminophen plus FeSO4; however, FeSO4 alone increased the intensity of staining of the immunoblot for protein aldehydes over control at all times after 0 time. Acetaminophen-protein adducts were detected in acetaminophen- and acetaminophen plus FeSO4-treated mice. In vitro experiments indicated that FeSO4 plus tert-butyl hydroperoxide in the presence of bovine serum albumin increased protein aldehyde formation. Inclusion of acetaminophen in the incubation mixture inhibited protein oxidation of bovine serum albumin in a concentration dependent manner. The data indicate that acetaminophen quenches protein oxidation, presumably by reacting with the hydroxyl radical. These data are consistent with the theory that acetaminophen covalent binding is the primary mechanism of toxicity and argue against a role for protein oxidation in acetaminophen hepatotoxicity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.